Normal Hematopoietic Stem Cell Function Research Articles

Efficacy Evaluation of a Tetracyclic Triterpene Compound Targeting Multiple Apoptosis Proteins for the Development of a Multiple Myeloma Treatment

Introduction:Multiple myeloma (MM) is an incurable hematologic cancer that originates in plasma cells and occurs primarily in older patients over 60. This study screened for hit compounds for MM treatments targeting mitochondria and apoptosis proteins. Further, non-clinical testing, including molecular docking analysis, was performed. Methods: Based on our previous studies, candidate compounds targeting mitochondria were screened (www.medchemexpress.com). The killing effect of the compounds on MM cell lines (RPMI8226, IM9, and U266) was examined using a sulforhodamine B assay. Apoptosis mechanisms were investigated in vitro. Complex immunocompromised mice were established by injecting RPMI8226-RFP-FLuc cells through the tail vein. Efficacy of the KBB-N2 activity was assessed following intraperitoneal (15 or 30 mg/kg/day) and tail vein (7 mg/kg/day) infusions for three days a week at daily intervals. Toxicity, pharmacokinetics, and molecular docking analyses were performed according to the established protocols. Results: The hit compound, KBB-N2, a tetracyclic triterpene, was identified as an MM-targeted compound with cytotoxic effects. The primary mechanism of action of KBB-N2 in MM cells was inhibiting the activity of the mitochondrial electron transport (ETC) complex, resulting in the generation of excess intracellular reactive oxygen species (ROS), which led to tumor cell death by ROS-mediated apoptosis. In addition, exposure to KBB-N2 inhibited the activity of catalase, a powerful antioxidant enzyme in MM cells. Severe suppression of normal hematopoietic stem cell function was observed with lenalidomide administration at a concentration of 25 μM. However, this toxicity was not observed when KBB-N2 was administered at doses up to 60 μM. KBB-N2 activity involves PARP cleavage and caspase activation in the apoptotic pathway. Furthermore, KBB-N2 inhibited cell growth and exerted cytotoxicity. KBB-N2 induced apoptosis by activating the P53 apoptosis pathway in MM cells. In vivo, all untreated tumor-bearing mice showed rapid tumor growth and severe plasmacytomas, which led to death within seven weeks. Mice treated with KBB-N2 had significantly inhibited tumor growth and longer survival times. The molecular docking assay revealed the optimal docking conformation binding energy of KBB-N2 with docking scores (kcal/M) of -7.3 and -9.8 for ETC complexes I and III, respectively. Conclusions: The triterpene compound, KBB-N2, was identified as a hit compound and may act as a targeted agent for MM treatment. The antimyeloma activity of KBB-N2 involves interference with the ETC complex and leads to MM cell death.

Simultaneous inhibition of Sirtuin 3 and cholesterol homeostasis targets acute myeloid leukemia stem cells by perturbing fatty acid β-oxidation and inducing lipotoxicity.

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease initiating leukemia stem cells (LSCs). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSCs. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSCs. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSCs but is not essential for normal human hematopoietic stem and progenitor cell (HSPC) function. To elucidate the molecular mechanisms by which SIRT3 is essential in LSCs we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support oxidative phosphorylation and ATP production in human LSCs. Further, we discovered two approaches to further sensitize LSCs to SIRT3 inhibition. First, we found that LSCs tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSCs to YC8-02 and potentiates LSC cell death. Second, SIRT3 inhibition sensitizes LSCs to BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.

Abstract 525: Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells

Abstract Therapies for acute myeloid leukemia (AML) patients are suboptimal due mainly to relapse of disease after treatment. Leukemia stem cell (LSC) populations maintain the disease and serve as a reservoir for relapse. Therefore, treatments that can eradicate LSCs will improve the outcomes of patients with AML. Our preliminary data have shown that the interleukin-1 (IL-1) pathway is upregulated following treatment and disease progression in AML and plays an important role in LSCs in vitro and in vivo. To target IL-1 signaling, we disclose the structure-activity relationship guided development of UR241-2, a novel small molecule compound of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4), which are critical downstream factors in IL-1/Toll-Like Receptor (TLR) signaling. UR241-2, at single dose (5 nM) screening exhibited high target selectivity; other than IRAK1/4 only 9 kinases in a panel of 682 kinases were inhibited by >25% activity. The NanoBRET assay using HEK293 cells transfected with targeted kinase vectors showed notably selective target engagement of UR241-2 against IRAK4 (IC50 = 6.07 nM). By using the in vitro NF-kB reporter assay and immunoblotting analysis, we identified that UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB and the phosphorylation of P65 and P38, following IL-1 stimulation. Next, we treated murine MLL-AF9 AML cells and human MDSL cells with UR241-2 and two IRAK4 inhibitors (PF-06650833 and CA-4948) that are currently in clinical or preclinical studies. We found that UR241-2 significantly inhibits colony-forming ability in AML and myelodysplastic syndromes, with similar or better efficacy compared to PF-06650833 or CA-4948. We also determined that UR241-2 suppresses LSC clonogenicity in primary human AML cells at diagnosis and in relapse while minimally impacting normal hematopoietic stem and progenitor cell function. Last, we investigated the impacts of UR241-2 on LSC engrafted ability in vivo. We treated murine MLL-AF9 cells with UR241-2 and PF-06650833 ex vivo for three days and transplanted them into recipient mice. After four weeks, we observed that UR241-2 significantly suppresses the engraftment levels of AML cells in bone marrow. It also decreases the WBC in peripheral blood and increases the RBC, HGB and PLT counts compared to the vehicle control group. Taken together, our findings demonstrated that targeting IRAK1/4 using the novel compound UR241-2 is able to impair LSC clonogenicity and engraftment capability in AML following treatment and disease progression. This provides a potential therapeutic strategy to eradicate the LSC population and improve the outcome of AML patients. Citation Format: Tzu-Chieh Ho, Rakesh K. Singh, Hiroki Kawano, Mark W. LaMere, Jian Wang, Paula M. Vertino, Nikolay V. Dokholyan, Laura M. Calvi, Craig T. Jordan, Michael W. Becker. Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 525.

Characterization of the Human Mesenchymal Stromal Compartment in Normal Homeostasis and Acute Myeloid Leukemia at the Level of the Single Cell

Mesenchymal stromal cells (MSCs) constitute a pivotal component of the bone marrow niche, providing key regulatory signals required for maintenance of normal hematopoietic stem cell (HSC) function. In recent years, there has been an increased appreciation of the role played by the bone marrow niche in leukemogenesis with increasing evidence pointing towards dysregulation of MSCs in acute myeloid leukemia (AML). However, studies to date have predominantly evaluated culture expanded MSCs which do not necessarily reflect the state of MSCs in their in situ milieu. This is further compounded by limitations inherent to bulk population measurements which fall short of capturing the underlying heterogeneity that may exist among seemingly identical cells. Leveraging the combined powers of single cell RNA sequencing (scRNA-seq) and mass cytometry (CyTOF), we provide here an in-depth characterization of the human bone marrow MSC compartment both in normal homeostasis and AML. We address the question of MSC heterogeneity, and through integrated analysis of our scRNA-seq data and recently published publicly available datasets, we show that MSCs in their native state are transcriptionally split into subsets of varying differentiation potential that exist along a continuum of cellular states. These subsets consist of a predominant cluster showing high expression of genes implicated in adipogenesis and a smaller cluster demonstrating upregulation of osteogenesis-related genes, suggesting that these cells are poised toward adipogenic and osteogenic differentiation respectively. In addition, we observe a much smaller population with a gene expression profile in keeping with that of more terminally differentiated osteoblasts, however this is not consistently observed across all samples. Furthermore, our comparative analysis of publicly available scRNA-seq datasets of murine stroma shows a high degree of conservation with similar patterns of cellular diversity. This pattern of cellular architecture is however lost in culture-expanded MSCs, underscoring the limitations associated with their use. In the context of AML, we show that native MSCs undergo transcriptional transformation, acquiring a distinct gene signature pattern distinguishing them from their normal counterparts. Among these changes are upregulation of genes implicated in inflammation indicating that deregulation of inflammatory signalling in AML is facilitated, at least in part, by cell-extrinsic mechanisms. In summary, our data reveal novels insights into the cellular architecture of the human MSC compartment in its native, unmanipulated state as well as unravel key transcriptional changes that characterize MSCs in AML, which can potentially be exploited for therapeutic targeting.

The Role of Stathmin-1 in the Regulation of Hematopoietic Stem Cells

Stathmin-1 is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of alpha-beta tubulin heterodimers. Also known as Oncoprotein 18 (OP18), Stathmin-1 is highly expressed in a wide variety of tumors, including high-risk myelodysplastic syndromes and acute leukemias (Machado-Neto et al, 2014). Stathmin-1 is also highly expressed in normal hematopoietic stem cells (HSCs), and its loss is associated with impaired erythroid differentiation and altered megakaryopoiesis (Ramlogan-Steel et al, 2021). While these prior studies have established a role of Stathmin-1 in normal erythro-megakaryopoiesis, its role in hematopoiesis- particularly in hematopoietic stem and progenitor cells- is not clear. Given the high expression of Stathmin-1 in HSCs and the importance of microtubule dynamics to HSC differentiation, we hypothesized that Stathmin-1 loss may impact normal HSC function. To address the role of Stathmin-1 in HSCs, we characterized HSC phenotype and function in a Stathmin-1 knockout mouse (Stmn1-/-). Under homeostatic conditions, Stmn1-/- HSC number and frequency are normal compared to wild-type (WT) mice. However, Stmn1-/- HSCs have markedly impaired function, as their bone marrow (BM) failed to engraft in competitive transplants (10% vs. 60% chimerism at 24 weeks post-transplant p<0.0001). Competitive intra-tibia transplants were then done to circumvent potential homing defects, and again Stmn1-/- BM displayed a significant engraftment defect compared to WT (10% vs. 78% chimerism at 24 weeks p<0.0001). Of note, Stmn-1-/- HSCs performed equivalently to WT HSCs in an in vivo homing assay, further suggesting their lack of engraftment is not due to homing defects. Stmn1-/- BM cells were capable of engraftment in non-competitive transplants, albeit less robustly than WT cells (42% vs. 90% p<0.01). Treatment of WT and Stmn1-/- mice with one single dose of the chemotherapeutic agent 5-fluorouracil showed that Stmn1-/-mice have slower tri-lineage recovery than their WT littermates (p<0.01 at day 15). Consistent with their reduced in vivo function, Stmn1-/- HSCs produced fewer colonies than WT HSCs when plated in Methocult. They were also not capable of serial replating, supporting that loss of Stathmin-1 is associated with impaired HSC function. Consistent with its known role in regulating microtubules, Stmn1-/- HSCs displayed altered microtubule morphology by confocal microscopy with a lack of normal nuclear invaginations compared to WT HSCs. Analysis of cycling status revealed that Stmn1-/- HSCs are more quiescent than WT HSCs at baseline (1.3% vs. 3.4% at S/G2/M phase) and in response to 24 hours of G-CSF stimulation (3.4% vs. 7% at S/G2/M phase). In addition, Stmn1-/- HSCs have higher rates of apoptosis than their WT counterparts (18.5% vs. 9.90%), suggesting that Sthamin-1 loss leads to both impaired HSC cycling and increased cell death. We next performed RNA-seq on sorted Stmn1-/- and WT HSCs to gain further mechanistic insight into their impaired function. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function, and cell differentiation in the Stmn1-/- HSCs compared to WT controls. Consistent with these results, measurement of mitochondrial function (oxygen consumption rate) using the Seahorse analyzer showed that Stmn1-/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT. Together, these data suggest a model whereby Stathmin-1 regulates microtubule dynamics in HSCs to promote optimal mitochondrial function to support cellular cycling, survival and self-renewal.

P424: INVESTIGATING GPR56 FUNCTION IN HUMAN IPSC-DERIVED HEMATOPOIETIC CELLS USING A TET-ON MODEL OF CONSTITUTIVE ACTIVATION

Background: GPR56 (ADGRG1) is a widely expressed adhesion G-coupled protein receptor in humans and mice which is implicated to play a role in both haematopoietic development and Acute Myeloid Leukaemia (AML). In AML, there exists a low frequency subpopulation of leukaemic stem cells (LSCs) which, similar to their benign Hematopoietic Stem Cell (HSC) counterparts, exhibit stem cell behaviours. High expression of GPR56 forms part of the molecular signature of these LSCs, and AML cases with high levels of GPR56 at diagnosis have poorer outcomes. Our hypothesis is that GPR56 activity underlies some of the stem cell behaviours seen in both HSCs and LSCs. Aims: To characterise the effects of GPR56 receptor activation so as to better understand its role in AML and hematopoietic development. Methods: We developed a Tet-On human inducible stem cell line (M1) transfected with a construct encoding a truncated GPR56 protein. This constitutively active GPR56 receptor form is overexpressed following doxycycline administration. Flow cytometry-based cell cycle and apoptosis studies were performed on the M1 line and wild type Sci55 (WT) iPSCs +/- doxycycline exposure. IncuCyte® transmigration studies utilising an SDF-α gradient were performed to examine cell migration. Finally, the M1 cells were subjected to a hematopoietic differentiation protocol and assessed for CD34+ CD38- compartment size at day 12 of differentiation. Results: GPR56 activation alters cell cycle progression with significant accumulation of M1 cells in the G0/1 and G2/M phases of the cell cycle compared to WT (p <0.005). GPR56 activation also causes a significant reduction in apoptotic cells in dox-treated M1 cells (p <0.005), despite doxycycline being pro-apoptotic in WT cells (p <0.05). GPR56 activation reduces the number of transmigrating M1 cells in response to an SDF-α gradient (p <0.05). Importantly, GPR56 activation during differentiation increases the CD34+ CD38- compartment size in M1 cells treated with doxycycline at day 12 (p<0.05) as compared to untreated cells Summary/Conclusion: GPR56 activation results in accumulation of undifferentiated M1 iPSCs in the G0/1 and G2/M phases, suggestive of cell cycle checkpoint arrest, and exerts a powerful anti-apoptotic effect. GPR56 activation also reduces cell transmigration in response to SDF-α, suggesting receptor involvement in cell adhesion and migration. Finally, GPR56 activation during hematopoietic differentiation results in an increased yield of CD34+ CD38- cells, suggesting that GPR56 activity may influence the proportion of immature cells arising during haematopoietic differentiation. These results are of interest in relation to normal HSC functions and may indicate the mechanisms through which overexpression of GPR56 (with increased activation) in LSCs contributes to poorer outcomes in AML. Future work will include identification and characterisation of the signalling pathways downstream of the GPR56 receptor to better understand this receptor’s role in the haematopoietic system in health and disease.

PLCG1 is required for AML1-ETO leukemia stem cell self-renewal

AbstractIn an effort to identify novel drugs targeting fusion-oncogene–induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

CXCL12-Expressing Bone Marrow Niches Differentially Regulate CML Compared to Normal Stem Cells

CXCL12 plays a major role in hematopoietic stem cells (HSC) localization to regulatory niches in the bone marrow microenvironment (BMM). Previous studies indicate that CXCL12 deletion from Prx+ mesenchymal stem cells (MSC) reduces murine HSC numbers, and from Tek+ endothelial cells (EC) modestly reduces their repopulating activity. In contrast, CXCL12 deletion from Ocn+ osteoblasts or Osx+ osteoprogenitors does not affect HSC function (Greenbaum et al. Nature. 2013 Mar 14;495(7440):227-30; Ding et al. Nature. 2013 Mar 14;495(7440):231-5.). These studies identified MSC and endothelial cells as important HSC niche components. However, niche cells for CML leukemic stem cells (LSC) remain poorly characterized. To evaluate the contribution of CXCL12-expressing populations to LSC regulation, we transplanted normal and CML BM cells (CD45.1/2+; 2*106/mouse) into CXCL12f/f (loxP sites flanking exon 2) mice crossed with Tek-Cre and Prx-Cre mice ( reported in ASH 2016, Blood, Abstract: 26) . We showed that CXCL12 deletion from Prx+ MSC resulted in reduction in normal HSC (LSK Flt3- CD150+ CD48- cells) numbers and function compared to controls, but did not observe significant reduction of HSC with CXCL12 deletion from Tek+ EC. Interestingly, transplantation of CML cells into mice with CXCL12 deleted from Prx+ MSC led to reduced survival compared to control mice associated with increased WBC counts, BM cellularity, LSC numbers, and secondary repopulation capacity. In contrast, CXCL12 deletion from Tek+ cells led to enhanced survival, associated with reduced BM cellularity and reduced LSC numbers. In further studies, we transplanted mixtures of equal numbers of normal and CML cells into Prx-Cre and Tek-Cre mice, and found a marked growth advantage of LSC compared with HSC in Prx-Cre mice, and a growth advantage of HSC over LSC in Tek-Cre knockout mice. We also show that both HSC and LSC demonstrate increased cell cycling via EdU labeling. Consistent with this, we found that Nilotinib treatment leads to significantly greater reduction in BM cellularity, LSC numbers and secondary repopulation potential in Prx-Cre mice, compared to Tek-Cre or WT mice. To evaluate the effects of CML on CXCL12-expressing BMM cells, we crossed CXCL12GFP (GFP reporter knocked into the CXCL12 locus) and SCL-tTA-BCR-ABL mice to generate CXCL12GFP-BCR-ABL mice. CML development lead to significantly reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) but increased GFP+ endothelial cells (CD45-Ter119-CD31+), and within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased compared to WT mice. To further understand niche mediated interactions that regulate LSC fate, we studied gene expression profiles of LSC from mice following Cre-mediated targeting of CXCL12, compared to LSC from Cre-negative mice. Our preliminary analysis indicates significant enrichment of genes associated with cell cycle progression and Myc activation, and negative enrichment of genes related to TGF-b, STAT3, and Polycomb Repressive Complex 2 (PRC2) activity in LSC from Prx1+Cre CXCL12f/f mice. Reduced TGF-b, STAT3 and PRC2/EZH2 activity represent potential mechanisms for regulation of CML LSC maintenance and/or TKI resistance, and may potentially contribute to Prx1+ MSC-mediated LSC regulation. We conclude that CXCL12+ BM MSC inhibit CML development, maintain LSC quiescence, and enhance TKI resistance, while CXCL12+ EC enhance LSC maintenance and disease development. Leukemia associated reduction in CXCL12+MSC and increase in CXCL12+EC may enhance CML development. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM MSC and EC for CML LSC compared with normal HSC. DisclosuresNo relevant conflicts of interest to declare.

Novel In Vivo Models Uncover Non-Cell-Autonomous Effects of Myelodysplastic Syndrome (MDS) on Marrow Osteolineage Cells and Hematopoietic Dysfunction

Myelodysplastic syndromes (MDS) remain a therapeutic challenge, leading to significant morbidity and mortality due to blood cytopenias and leukemic transformation. We and others have demonstrated that abnormalities of the bone marrow microenvironment (BMME) contribute to disease pathogenesis and progression. However, current models fail to assess in vivo the impact of the malignant cells on the BMME and on residual non-clonal hematopoiesis. We previously identified BMME abnormalities in Vav1-NUP98/HOXD13 (NHD13) mice, a well-established transgenic murine model that recapitulates pathologic hallmarks of human MDS including blood cytopenias, dysmyelopoiesis, and transformation. NHD13 mice had increased osteoblastic-lineage cells (OBC, lineage-/CD45-/CD31-/CD51+/Sca1-), as well as hematopoietic dysfunction and hematopoietic stem cell (HSC) loss.To determine if MDS can initiate BMME alterations, we transplanted MDS marrow of NHD13 mice or wild-type (WT) littermate marrow (CD45.2+/CD45.1+) into irradiated WT recipients (CD45.2+/CD45.1-). Using flow cytometry to distinguish donor- vs. host-derived hematopoietic cells, we found that NHD13 and WT donor-derived cells contributed to approximately 60% of recipient marrow, generating an in vivo model of MDS cells among genetically-WT hematopoietic cells. This transplant model recapitulates human MDS, where MDS clones emerge and expand among normal hematopoietic cells in a BMME that is initially genetically normal. At 6 weeks post-transplant, OBCs were increased in recipients of MDS marrow (p<0.05), indicating that interaction with MDS cells induced osteolineage cells to take on features of an MDS-BMME. Concurrent with BMME remodeling,HSCs were decreased in recipients of MDS marrowcompared to recipients of WT marrow (p<0.05). Notably, HSCs in recipients of MDS marrow were also decreased compared to surviving irradiated but non-transplanted mice (p<0.05), consistent with disruption of normal HSC function by MDS cells. In line with acquired hematopoietic dysfunction, recipients of MDS marrow exhibited leukopenia and anemia compared to recipients of WT marrow. To assess the hematopoietic function of genetically-WT recipient cells in the presence of MDS cells, we quantified donor- vs. recipient-derived peripheral white blood cells (WBC). The number of recipient-derived WBCs in mice transplanted with MDS marrow was lower compared to irradiated, non-transplanted mice. This suggests that cytopenias in recipients of MDS marrow were not only due to defective cell-autonomous hematopoiesis by MDS cells, but also due to the impaired hematopoietic function of residual WT recipient cells. Together, these data indicate that MDS cells may decrease hematopoietic support, leading to dysfunctional hematopoiesis.To assess hematopoietic function of non-irradiated WT hematopoietic cells in the presence and absence of MDS, we transplanted NHD13/GFP- marrow mixed 1:1 with WT/GFP+ marrow (NHD13/GFP-:WT/GFP+) or WT/GFP-:WT/GFP+ marrow into irradiated WT recipients. Although NHD13 donor-derived cells contributed to only 4% of reconstituted recipient marrow in this model, HSC depletion was unexpectedly observed in recipients of NHD13/GFP-:WT/GFP+ marrow compared to recipients of WT/GFP-:WT/GFP+ marrow, showing that, even when recipient marrow is comprised of over 90% normal hematopoietic cells, the HSC pool is reduced in the presence of MDS cells. These data demonstrate a strong non-cell-autonomous effect of MDS on hematopoietic function.Because human MDS is associated with diverse genetic abnormalities, we sought to determine if MDS-dependent BMME remodeling transcends the genetic makeup of MDS by assessing the BMME in mice with inducible EVI1 overexpression (EVI1++), an oncogenic transcription factor important in myeloid progenitor function and commonly implicated in human MDS. Marrow from EVI++ mice (mixed 1:1 with WT marrow) was transplanted into WT mice. By 14 weeks post-transplant, recipients of EVI1++ cells exhibited BMME alterations, demonstrated by increased OBCs (p<0.05) compared to recipients of WT cells. Thus, exposure to both NHD13 and EVI1++ marrow induces OBC remodeling in our transplant models.Together, these data uncover non-cell-autonomous effects of MDS causing hematopoietic dysfunction in vivo, and provide novel tools to discern the MDS signals and BMME changes impairing normal hematopoiesis. DisclosuresLiesveld:Seattle Genetics: Honoraria; Onconova: Honoraria.

Zebrafish models of acute leukemias: Current models and future directions.

Acute myeloid leukemias (AML) and acute lymphoid leukemias (ALL) are heterogenous diseases encompassing a wide array of genetic mutations with both loss and gain of function phenotypes. Ultimately, these both result in the clonal overgrowth of blast cells in the bone marrow, peripheral blood, and other tissues. As a consequence of this, normal hematopoietic stem cell function is severely hampered. Technologies allowing for the early detection of genetic alterations and understanding of these varied molecular pathologies have helped to advance our treatment regimens toward personalized targeted therapies. In spite of this, both AML and ALL continue to be a major cause of morbidity and mortality worldwide, in part because molecular therapies for the plethora of genetic abnormalities have not been developed. This underscores the current need for better model systems for therapy development. This article reviews the current zebrafish models of AML and ALL and discusses how novel gene editing tools can be implemented to generate better models of acute leukemias. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease Technologies > Perturbing Genes and Generating Modified Animals.

Potential of Sea-CD70 for the Treatment of Myeloid Leukemia

CD70 (CD27L) is a member of the tumor necrosis factor superfamily transiently expressed on activated adaptive and innate immune cells. Engagement of CD70 to the co-stimulatory receptor CD27, which is present on adaptive immune cells, promotes antigen-specific T cell responses. Both CD70 and CD27 have been reported to be aberrantly expressed on AML and MDS blasts, but are absent on normal hematopoietic progenitor cells, making them appealing therapeutic targets. CD70-CD27 interactions, working through auto- and/or paracrine signaling, can induce a stem-like signature and stimulate the proliferation of leukemic blasts. In addition, the CD70-CD27 interaction drives inflammatory cytokine production from both CD27 and CD70 pathways, resulting in cytokine production from leukemic blasts and the myeloid cells. Inflammatory signaling is a critical component of the crosstalk between hematopoietic stem and progenitor cells and their microenvironment during infection or hematopoietic stress. However, chronic inflammation can suppress normal hematopoietic stem cell function and eventually lead to bone marrow failure. Similarly, preleukemic and leukemic cell' interactions with the bone marrow microenvironment can induce secretion of pro-inflammatory factors, which in turn, could suppress normal blood cell formation. Therefore, blockade of the CD70-CD27 inflammatory signal pathway could help restore normal hematopoiesis, limit immune evasion, and increase the immune directed responses in MDS and AML. SEA-CD70 is a humanized, nonfucosylated monoclonal antibody targeting CD70. SEA-CD70 retains the ability to block CD27 mediated signaling and binds significantly better than the fucosylated parent to the FcγRIIIa receptor. SEA-CD70 mediates potent target killing through Fc-dependent antibody effector functions, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). SEA-CD70 can also disrupt CD70-CD27 signaling and could block cellular proliferation and survival of blast cells in vitro. SEA-CD70 may also inhibit the inflammatory signals originating from the interaction between the tumor microenvironment and leukemic blasts. In vivo, SEA-CD70 delayed tumor growth and increased survival in subcutaneous AML xenograft mouse tumor models both as a single agent and when combined with standard of care (SOC) agents. Overall, our data highlight the therapeutic potential of SEA-CD70 for the treatment of AML and MDS patients. Disclosures Diolaiti: Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Haass:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Ulrich:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Chen:Seattlke Genetics:Current Employment, Current equity holder in publicly-traded company.Ho:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Gardai:Seattle genetics:Current Employment, Current equity holder in publicly-traded company.

Abstract IA18: The PI3K isoforms in myeloid leukemia and hematopoietic stem cells

Abstract The Class IA PI3 kinase isoforms (p110α, β, and δ) transduce many growth factor signals that are important for the proliferation, differentiation, and self-renewal of hematopoietic stem cells (HSCs). Mutations in growth factor receptors or RAS proteins are commonly observed in patients with acute myeloid leukemia (AML), leading to activation of the PI3K/AKT pathway. Therefore, PI3K inhibition is an attractive therapeutic strategy for a large subset of AML patients. We previously reported that p110α is dispensable for HSC function, suggesting that redundancy exists between the Class IA isoforms in HSCs (Gritsman et al., J Clin Invest 2014). However, we have identified a specific role for p110α in RAS-mutated myeloid leukemia. Furthermore, we found that the p110α-selective inhibitor BYL-719 can sensitize RAS-mutated leukemic cells to the MEK inhibitor MEK-162. While PI3K inhibitors have multiple clinical indications, including in hematologic malignancies, it is still unclear whether PI3K plays an important role in normal HSC function. Upon environmental stresses like chemotherapy or infection, quiescent HSCs must enter the cell cycle to re-establish homeostasis and promote emergency myelopoiesis. To test for redundancy between p110α and p110δ in HSCs, we generated p110α and p110δ double knockout (DKO) mice, with germline deletion of p110δ and conditional deletion of p110α. DKO mice have leukopenia, anemia, and decreased numbers of lymphoid-primed multipotent progenitors (LMPPs) in the bone marrow, though HSC numbers are unchanged. In competitive repopulation assays, B-cell maintenance was severely impaired, while the myeloid and T-cell lineages were relatively preserved. This suggests that HSC function is not impaired at steady state. We performed transcriptome analysis of DKO, p110δ, and p110α KO HSCs and LMPPs after bone marrow transplantation. Gene set enrichment analysis revealed downregulation of gene sets associated with cell cycle progression. Interestingly, we also observed negative enrichment of inflammatory response gene sets in both p110δ KO and DKO HSCs and LMPPs. To examine the roles of p110δ and α in the hematopoietic stress response, we injected p110δ, DKO, and WT mice with 5-fluorouracil. We observed significantly decreased survival of 5-FU-treated DKO mice due to impaired hematopoietic recovery, associated with the failure of HSCs to enter the cell cycle. Consistent with this finding, we also observed a decrease in myeloid reconstitution by DKO HSCs in secondary competitive transplantation, suggesting that p110α and p110δ play redundant roles in emergency myelopoiesis. We also observed defects in downstream signaling in DKO HSCs and progenitors in response to IL1β or TNFα stimulation. Our results suggest that p110α and δ act in a redundant fashion to transduce specific inflammatory signals in HSPCs in response to hematopoietic stress. Our results have important implications for the use of PI3K inhibitors in combination with chemotherapy and in other hematopoietic stress conditions. Citation Format: Shayda Hemmati, Taneisha Sinclair, Meng Tong, Boris Bartholdy, Rachel Okabe, Kristina Ames, Leanne Ostrodka, Tamanna Haque, Imit Kaur, Anupriya Agarwal, Jean Zhao, Thomas Roberts, Kira Gritsman. The PI3K isoforms in myeloid leukemia and hematopoietic stem cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA18.

3147 – SPHINGOSINE-1-PHOSPHATE RECEPTOR 3 SIGNALING DEPLETES LEUKEMIA STEM CELLS BY INDUCING DIFFERENTIATION

Inflammation underlies many chronic diseases of aging, involves dysregulated myeloid cells, and has emerged as a critical process regulating both normal hematopoietic stem cell (HSC) function and myeloid malignancy. However, few of the signaling pathways that underlie these proposed linkages are known. Here, we show that bioactive lipid signaling through sphingosine 1-phosphate receptor 3 (S1PR3) regulates human myeloid lineage determination via inflammatory programs and is dysregulated in acute myeloid leukemia (AML). S1PR3 is myeloid-restricted and not expressed by quiescent HSC, but upon overexpression (OE) promoted myelopoiesis and activated inflammatory signatures. S1PR3 is deficient in primitive AML, but enriched in more mature cases. Importantly, in functionally-validated primary leukemia stem cells (LSC) sorted for high S1PR3 expression or when differentiation was induced through S1PR3OE exhibited reduced xenograft repopulating ability. Treatment of recipient mice engrafted with the S1P prodrug FTY720 reduced patient AML LSC frequency. Thus, S1PR3 marks a subset of less primitive AML cells with a distinct inflammatory signature, and activation of S1PR3 may represent a novel therapeutic approach to disrupt LSC function through induction of differentiation.

The Role of Deubiquitinating Enzymes in Hematopoiesis and Hematological Malignancies.

Hematopoietic stem cells (HSCs) are responsible for the production of blood cells throughout the human lifespan. Single HSCs can give rise to at least eight distinct blood-cell lineages. Together, hematopoiesis, erythropoiesis, and angiogenesis coordinate several biological processes, i.e., cellular interactions during development and proliferation, guided migration, lineage programming, and reprogramming by transcription factors. Any dysregulation of these processes can result in hematological disorders and/or malignancies. Several studies of the molecular mechanisms governing HSC maintenance have demonstrated that protein regulation by the ubiquitin proteasomal pathway is crucial for normal HSC function. Recent studies have shown that reversal of ubiquitination by deubiquitinating enzymes (DUBs) plays an equally important role in hematopoiesis; however, information regarding the biological function of DUBs is limited. In this review, we focus on recent discoveries about the physiological roles of DUBs in hematopoiesis, erythropoiesis, and angiogenesis and discuss the DUBs associated with common hematological disorders and malignancies, which are potential therapeutic drug targets.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

SummaryHematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

Functional Impact of MacroH2A Histone Variants in Acute Myeloid Leukemia

Histone variants are emerging as key regulatory molecules in cancer. Recent data provided first evidence of how transcriptional deregulation and changes in the deposition of histone variants may affect malignant transformation. H2A variants are highly conserved and consist of 8 members. Among all histone variants, macroH2A variants hold a unique C-terminal macro domain, which is approximately twice the size of its H2A-like histone domain. In mammals, there are 3 macroH2A variants encoded by H2AFY (macroH2A1.1, macroH2A1.2) and H2AFY2 (macroH2A2). MacroH2A variants occupy large repressive domains throughout the genome, exerting a repressive role on transcription. So far, the function of macroH2A proteins in hematopoiesis and leukemic transformation is incompletely understood. Here, we report on a functional role of macroH2A histone variants that may influence cell competition in leukemia and normal hematopoietic stem and progenitor cell (HSPC) function. In published gene expression datasets, macroH2A variants H2AFY and H2AFY2 are highly expressed in hematopoietic stem cells (HSCs) compared to mononuclear cells (both p&lt;0.0001****). Moreover, expression of H2AFY and H2AFY2 appeared even higher in acute myeloid leukemia (AML) (p=0.0750 and 0.0037**, respectively) when compared to the expression level in HSCs. This high expression is independent of different genetic subtypes and AML risk groups. To assess for the functional dependency of leukemic cells on the expression of macroH2A variants, we performed an in vitro CRISPR-Cas9 dropout screen focusing on macroH2A variants in MOLM-13 cells. Genetic inactivation of H2AFY but not H2AFY2 resulted in outcompetition of infected cells against non-infected competitors. This finding could be recapitulated in 3 additional AML cell lines (OCI-AML3, THP-1, HL-60), indicating a broader dependency on H2AFY irrespective of the oncogenic background or underlying driver mutation. To validate the functional impact of H2AFY on leukemia development in vivo, we generated a novel conditional knockout mouse model for H2afy and induced leukemia through retroviral infection of sorted Lineage-Sca1+cKit+ (LSK) cells with the oncogene MLL-AF9. Results of these functional studies will be presented. To understand the functional impact of macroH2A variants on normal HSPC function in vivo, we performed competitive repopulation studies. We used conventional knockout mouse models for the 2 H2afy isoforms: macroH2A1.1 and macroH2A1.2 In addition, for inactivation of both isoforms, we used the conditional macroH2A knockout mouse model. H2afy2 was either inactivated by RNAi or conditionally knockout through Mx1-Cre recombinase activation. In brief, whole bone marrow cells (WBMCs) were transplanted into primary recipient mice in a 1:1 competitive manner and compared to the respective wildtype littermate controls. Peripheral blood (PB) chimerism was monitored on a monthly basis 4 weeks post-transplantation for a total of 16 weeks. We observed no significant difference in the recipients of macroH2A1.1 (n=5/5) or macroH2A1.2 (n=10/9), suggesting both macroH2A isoforms are dispensable for intrinsic functions of HSCs. Remarkably, we found macroH2A2-deficient cells (n=6) competed significantly worse than macroH2A2+/+ (n=5) controls in PB (p=0.0043**) and in WBM (p=0.0043**), indicating a requirement for macroH2A2 in maintenance of HSPCs. These findings could be recapitulated by RNA-mediated inactivation of H2afy2 in sorted HSCs. We observed significant reduction of macroH2A2-depleted hematopoiesis after 16 weeks in WBM (p=0.0286*) and PB (p=0.0079**). Importantly, this functional impairment was less pronounced when investigating multipotent progenitor and committed progenitor cell function in spleen colony-forming unit (CFU) assay in vivo or CFU assay in vitro. Taken together, our data indicate distinct requirements for macrohistone variants in leukemic and normal hematopoietic cells. Dependency of leukemic cells but not normal HSPCs on macroH2A may indicate a potential therapeutic index. Disclosures No relevant conflicts of interest to declare.

SETDB1 Mediated H3K9 Methylation Suppresses MLL-Fusion Target Expression and Leukemic Transformation

Genes encoding epigenetic proteins are mutated in about 70% of Acute Myeloid Leukemia patients underscoring their role in leukemic transformation. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), such as those harboring MLL rearrangements, NPM mutations and others. Indeed, expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow cells into a lethal AML. We have shown that epigenetic regulation of Hoxa9 and Meis1 is mediated through the Polymerase Associated Factor 1 complex (PAF1c). The PAF1c binds to RNA polymerase II and recruits enzymes that alter epigenetic landscapes and influence gene transcription. We and others have shown that the PAF1c directly binds and recruits MLL and MLL fusion proteins to target genes such as Hoxa9 and Meis1 to deliver H3K4 methylation or H3K79 methylation activity respectively. Importantly, the PAF1c-MLL1 interaction is necessary for the growth of MLL-fusion leukemia cells but appears dispensable for normal hematopoietic stem and progenitor cells (HSPCs) function. This suggests the PAF1c is differentially required in leukemic cells compared to its normal counterparts. Thus, we aimed to 1) define the role of the PAF1c in normal hematopoiesis, and 2) identify mechanisms regulating PAF1c function that may be deregulated in leukemia. To understand the role of the PAF1c in hematopoiesis, we conditionally deleted the PAF1c subunit Cdc73 in HSPCs in hematopoietic cells. Conditional hematopoietic deletion of Cdc73 leads to lethality within a week due to a depletion of c-kit+ cells. Characterization revealed a cell autonomous requirement for Cdc73 to prevent apoptosis in HSPCs. Gene expression profiling on both c-kit+ hematopoietic cells and AML cells revealed that Hoxa9/Meis1 gene programs are differentially responsive to loss of Cdc73. This work revealed an essential role for the PAF1c subunit in maintaining viability of HSPCs and points to AML specific functions for the PAF1c. Thus, we utilized immunoprecipitation followed by mass spectroscopy to identify novel PAF1c interactions in AML cells. We found a novel interaction with several H3K9 methyltransferases including SETDB1. We demonstrate that the PAF1c-SETDB1 interaction reduces expression of Hoxa9 and Meis1 by promoting deposition of H3K9me3. These findings mirrored human AML patient samples that showed SETDB1 expression was inversely correlated with HOXA9 and MEIS1 expression. Further, SETDB1 expression is correlated to disease status and overall survival in AML patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of AML cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While AML cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of HSPCs. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of AML cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with AML, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in AML that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML. Disclosures Maillard: Genentech: Consultancy; Regeneron: Consultancy.

Combination Treatment with Imetelstat, a Telomerase Inhibitor, and Ruxolitinib Depletes Myelofibrosis Hematopoietic Stem Cells and Progenitor Cells

Therapy with imetelstat (Ime), a telomerase inhibitor, has been shown to have disease-modifying effect in patients with myelofibrosis (MF) (Tefferi N Engl J Med 2015; Mascarenhas Blood 2018). This has been attributed to the ability of Ime to selectively deplete MF hematopoietic stem cells (HSC)/progenitor cells (HPC) (Wang Blood Adv 2018). As reported, the primary toxicities of Ime are cytopenias, however, they appear to be reversible, and most importantly, manageable without meaningful clinical consequences (Mascarenhas Blood 2018, Fenaux EHA 2019). Ruxolitinib (Rux) is the first and only JAK 1/2 inhibitor approved for use in patients with intermediate- or high-risk MF. Based on different mechanisms of action for Ime and Rux, we hypothesize that Ime in combination with Rux might create a regimen that would be more efficacious than single agent alone in depleting MF HSCs/HPCs. Using in vitro HPC assays and in vivo HSC assays, we evaluated the therapeutic potential of Rux and Ime. MF splenic (n=7) or cord blood (CB) (n=3) CD34+ cells (2.5×104/mL) were incubated in serum free expansion medium containing cytokines in the presence of Rux (50nM) alone, Ime alone (1.8uM), Rux+Ime (simultaneous treatment) or Rux→Ime (sequential treatment). Rux was dosed for 3 days and Ime for 14 days under treatment conditions containing either or both drugs. In addition, parallel cultures with mismatched oligonucleotides (MM) or vehicle alone were performed. Compared to either drug alone or to MM control, Rux followed by Ime sequential treatment resulted in significant reductions in the numbers of MF Lin-CD34+ cells (Rux→Ime vs. Rux alone, p=0.001; vs. Ime alone, p=0.059; vs. Rux→MM, p=0.052) and assayable HPCs [(CFUGM+BFU-E+CFU-GEMM), Rux→Ime vs. Rux alone, p=0.02; vs. Ime alone, p=0.05; vs. Rux→MM, p=0.04], while the simultaneous treatment with Rux+Ime did not significantly reduce MF Lin-CD34+ cells and assayable HPCs. By contrast, none of these treatment conditions affected the behavior of normal HSCs/HPCs. Genotyping of individual colonies showed that sequential drug treatment of CD34+ cells from 2 JAK2V617+ MF patients resulted in greater reductions in both the percentage and absolute numbers of JAK2V617F+ myeloid progenitors than that achieved with Ime alone or simultaneous combination treatment. These findings suggest that an additive inhibitory activity against MF HSCs and malignant HPCs can be achieved with sequential treatment of Rux followed by Ime. We next assessed if treatment with Ime (10mg/kg, 3 times/week for 4 weeks, I.P. injection) and Rux (45mg/kg, daily for 7 days, oral gavage) had additive effects on MF or normal HSCs by directly treating NSG mice transplanted with MF or CB CD34+ cells with these two drugs either alone, in combination simultaneously or sequentially. Mice were sacrificed 4 months after the transplantation. Both simultaneous and sequential drug treatment of mice transplanted with MF splenic CD34+ cells resulted in greater reductions in the degree of human CD45+ marrow cell chimerism (relative to vehicle alone: Rux→Ime: 54.0%; Rux+Ime: 72.4%; Rux alone: 76.6%, Ime alone: 80.0%) and human CD45+ splenic cell chimerism (relative to vehicle alone: Rux→Ime: 13.8%; Rux+Ime: 31.9%; Rux alone: 163.1%; Ime alone: 42.4%) as compared with either drug alone treatment. Similar to our findings in the above in vitro studies, a greater reduction in the absolute number of human CD34+ cells was observed with sequential but not simultaneous combination treatment as compared with either drug alone treatment in the marrow of the mice transplanted with MF CD34+ cells (relative to vehicle alone: Rux→Ime: 36.0%; Rux+Ime: 61.0%; Rux alone: 47.5%, Ime alone: 57.3%). Furthermore, similar results were also observed in depletion of MF long-term HSCs in mice receiving splenic CD34+ cells from an additional patient. However, this same sequential drug schema did not affect normal HSC function. Collectively, these data indicate that alterations of scheduling of the administration of Rux and Ime affect the efficacy of this drug combination in depleting MF HPC/HSCs. We propose that cycles of Rux followed by Ime represents a potentially effective therapeutic strategy that is capable of eliminating MF HSCs/HPCs with an acceptable toxicity profile. Disclosures Hoffman: Merus: Research Funding.

Targeting the RNA m6A Reader YTHDF2 Selectively Compromises Cancer Stem Cells in Acute Myeloid Leukemia.

SummaryAcute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.

USP44 is dispensable for normal hematopoietic stem cell function, lymphocyte development, and B-cell-mediated immune response in a mouse model

Ubiquitin-specific protease 44 (USP44) is a nuclear protein with deubiquitinase (DUB) catalytic activity that has been implicated as an important regulator of cell cycle progression, gene expression, and genomic stability. Dysregulation in the molecular machinery controlling cell proliferation, gene expression, and genomic stability in human or mouse is commonly linked to hematopoietic dysfunction, immunodeficiency, and cancer. We therefore set out to explore the role of USP44 in hematopoietic and immune systems through characterization of a Usp44-deficient mouse model. We report that USP44 is dispensable for the maintenance of hematopoietic stem cell numbers and function under homeostatic conditions, and also after irradiation or serial transplantation. USP44 is also not required for normal lymphocyte development. Usp44-deficient B cells show normal activation, proliferation, and immunoglobulin class switching in response to in vitro stimulation, and Usp44-deficient mice mount normal antibody response to immunization. We also tested the effects of USP44 deficiency on disease progression and survival in the Emu-myc model of mouse B-cell lymphoma and observed a trend toward earlier lethality of Usp44-/- Emu-myc mice; however, this did not reach statistical significance. Overall, we conclude that USP44 is dispensable for the normal physiology of hematopoietic and immune systems, and its functions in these systems are likely redundant with other USP family proteins.